Background: Sos1 and Sos2 are guanine-nucleotide exchange factors for Ras and Rac small GTPases, which are involved in a wide range of cellular responses including proliferation and migration. We have previously shown that Sos1 and Sos2 have different effects on cell migration, but the underlying mechanisms are not clear. Methods: Using a 4-hydroxytamoxifen-inducible conditional Sos1KO mutation, here we evaluated the functional specificity or redundancy of Sos1 and Sos2 regarding the control of cell migration and dynamics of focal adhesions (FAs) in primary mouse embryonic fibroblasts (MEFs). Results: Functional analysis of the transcriptome of primary Sos1/2WT, Sos1KO, Sos2KO and Sos1/2DKO-MEFs revealed a specific, dominant role of Sos1 over Sos2 in transcriptional regulation. Sos1KO MEFs had an increased number and stability of focal adhesions (FAs) and curbed protrusion and spreading. Conversely, Sos2KO MEFs displayed unstable FAs with increased protrusion. Interestingly, Sos1, but not Sos2, ablation reduced the levels of GTP-bound Rac at the leading edge. In 3D, however, only Sos1/2KO MEFs showed increased invasion and matrix degradative capacity, which correlated with increased expression of the Mmp2 and Mmp9 gelatinases. Moreover, increased matrix degradation in Sos1/2KO MEFs was abrogated by treatment with Mmp2/9 inhibitors. Conclusions: Our data demonstrate that Sos1 and Sos2 have different functions in FAs distribution and dynamics in 2D whereas in 3D they act together to regulate invasion and unveil a previously undescribed mechanistic connection between Sos1/2 and the regulation of Mmp2/9 expression in primary MEFs.